Immunoglobulins (antibodies) in adult humans are categorized into five different isotypes: IgA, IgD, IgE, IgG, and IgM. The isotypes vary in size and sequence. On average, each immunoglobulin has a molecular weight of about 150 kDa. It is well known that each immunoglobulin comprises two heavy chains (H) and two light chains (L), which are arranged to form a Y-shaped molecule. The Y-shape can be conceptually divided into the Fab region, which represents the top portion of the Y-shaped molecule, and the Fc region, which represents the bottom portion of the Y-shaped molecule.
The heavy chains in IgG, IgA, and IgD each have a variable domain (VH) at one end followed by three constant domains: CH1, CH2, and CH3. The CH1 and CH2 regions are joined by a distinct hinge region. A CH2 domain may or may not include the hinge region. The heavy chains in IgM and IgE each have a variable domain (VH) at one end followed by four constant domains: CH1, CH2, CH3, and CH4. Sequences of the variable domains vary, but the constant domains are generally conserved among all antibodies in the same isotype.
The Fab region of immunoglobulins contains the variable (V) domain and the CH1 domain; the Fc region of immunoglobulins contains the hinge region and the remaining constant domains, either CH2 and CH3 in IgG, IgA, and IgD, or CH2, CH3, and CH4 in IgM and IgE.
Target antigen specificity of the immunoglobulins is conferred by the paratope in the Fab region. Effector functions (e.g., complement activation, interaction with Fc receptors such as pro-inflammatory Fcγ receptors, binding to various immune cells such as phagocytes, lymphocytes, platelets, mast cells, and the like) of the immunoglobulins are conferred by the Fc region. The Fc region is also important for maintaining serum half-life. Serum half-life of an immunoglobulin is mediated by the binding of the Fc region to the neonatal receptor FcRn. The alpha domain is the portion of FcRn that interacts with the CH2 domain (and possibly CH3 domain) of IgG, and possibly IgA, and IgD or with the CH3 domain (and possibly CH4 domain) of IgM and IgE.
Examining the constant domains of the immunoglobulin heavy chains more closely, the CH3 domains of IgM and IgE are closely related to the CH2 domain in terms of sequence and function. Without wishing to limit the present invention to any theory or mechanism, it is believed that the CH2 domain (or the equivalent CH3 domain of IgM or IgE) is responsible for all or most of the interaction with Fc receptors (e.g., Fcγ receptors), and contains histidine (His) residues important for serum half-life maintenance. The CH2 domain (or the equivalent CH3 domain of IgM or IgE) also has binding sites for complement. The CH2/CH3 domain's retention of functional characteristics of the antibody from which it is derived (e.g., interaction with Fcγ receptors, binding sites for complement, solubility, stability/half-life, etc.) is discussed in Dimitrov (2009) mAbs 1:1-3 and Dimitrov (2009) mAbs 1:26-28 and Prabakaran et al. (2008, Biological Crystallography 64:1062-1067). Consequently, CH2 domains have been used as scaffolds as alternatives to full-length antibodies.
Without wishing to limit the present invention to any theory or mechanisms, it is believed that some modifications to the CH2 domain may have only small effects on the overall structure of the CH2 domain (or CH2-like domain), and it is likely that in cases where the modified CH2 structure was similar to the wild-type CH2 structure the modified CH2 domain would confer the same functional characteristics as the wild-type CH2 domain possessed in the full immunoglobulin molecule.
It is known that efficacy of a therapeutic antibody (or fragment thereof) can be limited by an immune reaction. To address such issues, many methods have been used to humanize antibodies derived from a non-human source with the aim of reducing the human anti-murine antibody (HAMA) response, for example. One such method includes CDR grafting wherein CDRs from a non-human antibody are transferred to a human antibody scaffold. This method, however, may result in a reduction in binding to the target antigen, which may be a consequence of the imperfect fit between the antibody scaffold and the CDRs that results in a loss in molecular recognition between the antigen and the “antibody.”
Some methods are used with the aim of preserving the surface recognition features of the antigen-antibody interface (Raghunathan, 2009). Rather than simply transferring a CDR amino acid sequence from one antigen binding molecule to replace a structural loop in another immunoglobulin scaffold, these methods take other characteristics of the antigen binding molecule being transferred into account to preserve the three dimensional orientation of the amino acids and their interactions with framework region amino acids. For example, when constructing a humanized antibody, human frameworks are selected based on sequence similarity of the non-human and human frameworks, length of the 3 “CDR” loops, and the sequence similarity of the loop residues.
The present invention features novel CH2 domain template molecules and methods of design of such CH2 domain templates wherein loops from a database of domains (the “donor loops”) are transferred to a CH2 domain scaffold (“the acceptor”). The donor loops may be chosen based on length, for example the chosen donor loop may have a length that is similar (but not necessarily identical) to that of a structural loop in the CH2 domain scaffold. The CH2 domain scaffold may be derived from a CH2 domain of human IgG or from a CH2 domain of a different Ig or from a CH2 domain of a different mammal, e.g., macaque.
The CH2 domain has a traditional Ig-fold with a 13 sheet sandwich comprising 3 pairs of β strands. A disulfide bond connects the middle 13 strands. The strands are denoted by A, B, C, D, E, F and G. Intervening loops (sometimes called structural loops) are denoted as BC, DE and FG. As used herein, loops BC, DE and FG will be referred to as L1, L2 and L3 respectively. These three loops bind to the Fc-Gamma receptor when present as part of the Fc dimer. The other three loops, AB, CD and EF bind to the Fc-Rn receptor when present as part of the Fc dimer. While the CH2 domain scaffold is broadly similar to that of an Ig domain, there are variations both in the sequence signatures and structure. One distinct difference in structure is the D strand. This region is a typical beta strand in most Ig domains, but it is a coil in the CH2 domain. This structural difference in the D region may have entropic effects on the L2 loop. The transfer of loops to the CH2 domain can have an effect on the binding and stability of the engineered molecule. Thus, the present invention is different from traditional methods of antibody engineering involving loop grafting (e.g., traditional humanizing of antibodies) and transferring a loop to a variable domain. Referring to the loop transfer from donor molecules to the CH2 domain scaffolds of the present invention, it is difficult to predict what would be a good loop match based on the amino acid sequence of a loop in a typical immunoglobulin antigen binding region (e.g., since there are significant differences in the sequence patterns and structure). The transfer of loops from a donor to an acceptor molecule would affect the binding and stability of the molecule.
In the present invention at least one or up to three loops (e.g., L1, L2, L3, L1 and L2, L1 and L3, L2 and L3, or L1 and L2 and L3) from a donor are transferred to the CH2 domain. Without wishing to limit the present invention to any theory or mechanism, we believe that a careful rational transfer of such compatible structural loops from a selected donor may ensure preservation of the stereochemistry and surface topology of the antigen binding region of the donor molecule. Also, we believe that preservation of interactions among the loops and between the loops and the proximal β strands may lead to molecules that have desirable biophysical and biochemical properties (e.g., stability, solubility). While we believe that compatible loops may help to maintain affinity with the target, we believe variations in loop lengths may provide recognition with different types of antigens.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description.